Microwave apparatus and methods for performing chemical reactions

ABSTRACT

The present invention relates to an apparatus and methods for performing chemical reactions. In particular, the present invention relates to an apparatus for heating chemical reaction mixtures. The apparatus applies one or more semiconductor based microwave generators making the apparatus suitable for parallel processing of chemical reaction mixtures. The invention further relates to methods for performing chemical reactions, e.g. methods for heating a plurality of samples simultaneously or sequentially, methods for monitoring a microwave heated chemical reaction, and methods where the optimum conditions with respect to parameters, such frequency and applied power can be determined for the system consisting of apparatus plus sample.

This application is a divisional Application of U.S. patent applicationSer. No. 10/775,072 filed on Feb. 11, 2004, presently pending, which isa divisional of U.S. patent application Ser. No. 10/147,997 filed May20, 2002, now U.S. Pat. No. 6,720,540, which is a continuation of U.S.patent application Ser. No. 09/857,455, filed Jun. 27, 2001, now U.S.Pat. No. 6,403,939, which is National Stage Entry of PCT/IB99/02021filed Dec. 17, 1999.

The present invention relates to an apparatus for heating chemicalreaction mixtures. In particular, the present invention relates to anapparatus applying one or more semiconductor based microwave generatorsmaking the apparatus suitable for parallel processing of chemicalreaction mixtures. The invention further relates to methods forperforming chemical reactions, e.g. methods for heating a plurality ofsamples simultaneously or sequentially, methods for monitoring amicrowave healed chemical reaction and methods where the optimumconditions with respect to frequency and applied power can bedetermined.

One of the major obstacles for an organic chemist today is the timeconsuming search for efficient routes in organic synthesis. As anexample, the average performance some ten years ago in thepharmaceutical industry was around 25-50 complete substances per chemistper year resulting in an equal amount of new chemical entities aspotential new drug candidates. Today the figure is several 100's peryear and will soon be expected to be in the region of 1000's per year.

Thus, the challenges for the pharmaceutical industries and the organicchemist include identification of ways of reducing time in drugdevelopment, identification of ways of creating chemical diversity,development of new synthesis routes and maybe reintroduction of old“impossible” synthetic routes. Also, it is a constant challenge to reachclasses of totally new chemical entities.

As it will be apparent from the following, microwaves assisted chemistryoffers a way to circumvent at least some of the above-mentionedproblems, namely

speeding up the reaction time with several orders of magnitudes,

improving the yield of chemical reactions,

offering higher purity of the resulting product due to rapid heating andthereby avoiding impurities from side reactions, and

performing reactions which are not possible with conventional thermalheating techniques.

Microwave assisted chemistry has been used for many years. However, theapparatuses and methods have to a great extent been based onconventional domestic microwave ovens. Domestic microwave ovens have amultimode cavity and the energy is applied at a fixed frequency at 915MHz or 2450 MHz (depending on country). The use of single mode cavitieshave also been reported, see e.g. U.S. Pat. No. 5,393,492 and U.S. Pat.No. 4,681,740.

The market for microwave generators is totally dominated by magnetrons.In some situations travelling wave tubes (TWT) are used to amplify amicrowave signal. There are several disadvantages related to theconventional apparatuses. Some of these will be listed in the following:

It is a disadvantage that the energy distribution in conventionalmicrowave ovens is non-uniform. This leads to a varying temperature inthe sample depending on the position of the sample in the oven.Furthermore, the non-uniform energy distribution makes it difficult toobtain reproducible results. This effect is especially noticeable if anarray of sample holders such as a microtiter plate (e.g. with 96 wells)is used. Rotation of the sample in the oven does not significantlyimprove the reproducibility.

In conventional systems the power provided to each sample in an array ofsamples can only be calculate as an average power per sample by dividingthe measured input power with the total number of samples. Due to thenon-uniform energy distribution in the cavity this calculation will onlyprovide a rough estimate of the applied power to each sample.

One way of controlling the reaction is to monitor pressure andtemperature in all individual wells. This may give information of theconditions in a specified well during a particular run. Changing theposition will give a different result leading to poor reproducibility.An alternative way of trying to obtain a uniform energy distribution isto place a large load in the cavity in order to absorb energy moreuniformly.

Single mode cavity resonators offer a possibility of high efficiency andcontrolled heating patterns in small loads. However, the dielectricproperties of the load often change considerably with temperature,resulting in very large variations in power absorption since anessentially constant frequency microwave generator is used. Hence, theprocess becomes difficult to predict.

A further disadvantage of conventional system relates to the fact thatmagnetrons usually only provide a fixed frequency or a minor adjustmentaround the centre frequency of the magnetron. Furthermore, magnetronshave an unpredictable behaviour and are extremely temperature sensitive,especially when the efficiency decreases, towards the end of its “life”.

TWT's have been used as variable frequency amplifiers. However, TWT'sare rather expensive and often very complicated to use. Furthermore,TWT's require warm-up time before start meaning that TWT's cannotrapidly be switched on and off. In addition, wear out of TWT's isassociated with high maintenance costs.

Both magnetrons and TWT's require a high voltage power supply, which isa disadvantage in view of complications and the risk.

In U.S. Pat. No. 5,521,360 a variable frequency heating apparatus forproviding microwaves into a furnace cavity is described. The apparatuscomprises a voltage controlled microwave generator, a voltage controlledpre-amplifier and a power amplifier. The power amplifier may be a TWT.The TWT is operationally connected to the furnace cavity. The powerdelivered to the furnace is determined by measuring the power reflectedfrom the furnace using a power meter. Upon placing a sample in thecavity furnace, power may be coupled to the sample causing thetemperature of the sample to change.

The system described in U.S. Pat. No. 5,521,360 suffers from theabove-mentioned disadvantages relating to e.g. TWT's.

It is a further disadvantage of the apparatus described in U.S. Pat. No.5,521,360 that it is restricted to be used with only one cavity furnace,i.e. parallel heating of a plurality of samples using different heatingparameters is not possible.

It is another object of the present invention to provide an apparatuscomprising a first semiconductor based electromagnetic generator, and afirst applicator for holding a sample, which apparatus are capable ofperforming a controlled heating of the sample.

It is another object of the present invention to provide an apparatuscapable of performing parallel processing of many samples, withindividually settings of process parameters such as frequency, power,temperature, pressure etc.

It is a further object of the present invention to provide an apparatuscapable of monitoring many samples in parallel, with individuallymonitoring of process parameters such as frequency, power, temperature,pressure etc.

It is a still further object of the present invention to provide anapparatus capable of controlling many samples in parallel, withindividually adjustments of process parameters such as frequency, power,temperature, pressure etc.

It is a still further object of the present invention to provide anapparatus in which samples can be evenly heated by using variousapplicators.

It is a still further object of the present invention to provide anapparatus in which the frequency of the applied energy can be varied.

It is another object of the present invention to provide an apparatuscomprising a first semiconductor based electromagnetic generator, and afirst applicator for holding a sample, which apparatus are capable ofperforming a controlled heating of the sample.

It is a still further object of the present invention to provide anapparatus in which it is possible to measure the temperature in thereaction vessel by monitoring the change in resonance frequency of asecond material introduced into the reaction chamber. This materialcould be a crystal, semiconductor or any other solid state material witha temperature dependent resonance frequency.

The above-mentioned objects are complied with by providing in a firstaspect an apparatus for providing electromagnetic radiation to a firstapplicator, said apparatus comprising:

a) a first generating means for generating electromagnetic radiation,

b) a first amplifying means for amplifying the generated electromagneticradiation,

c) means for guiding the amplified electromagnetic radiation to thefirst applicator, and

d) means for controlling the first generating means and the firstamplifying means,

wherein the generated electromagnetic radiation comprises a plurality offrequencies, and wherein the first generating means and the firstamplifying means are essentially constituted by semiconductorcomponents.

By essentially constituted by semiconductor components is meant that thecomponents generating the electromagnetic radiation—such as the requiredpower transistors—are semiconductor based power transistors.

In the present context, guiding means should be interpreted as any meanscapable of guiding electromagnetic radiation such as metallic channelsor cables, such as coaxial cables or waveguides. The guiding means mayalso comprise active and/or passive components such as couplers,dividers, splitters, combiners, isolators, power meters, artificialloads, spectrum analysers etc.

In order to perform parallel processing of a plurality samples theapparatus may comprise a second applicator and suitable guiding meansfor guiding at least part of the amplified electromagnetic radiation tothe second applicator. Generally it may be favourable to be able toprovide electromagnetic radiation with different frequencies to thefirst and second applicator. Therefore, the apparatus may comprise asecond generating means for generating electromagnetic radiation at aplurality of frequencies and a second amplifying means for amplifyingthe electromagnetic radiation generated by the second generating means.In order to provide electromagnetic radiation at a plurality offrequencies the second generating means and the second amplifying meansare preferably constituted by semiconductor components, such assemiconductor based power transistors. Examples of such powertransistors are silicon-carbide power transistors. It is evident thatthe same type of transistors may be used in first generating means andthe first amplifying means.

To increase flexibility of the apparatus, the guiding means may comprisemeans for guiding the electromagnetic radiation amplified by the secondamplifying means to the second applicator. In addition, the guidingmeans may further comprise means for guiding at least part of theelectromagnetic radiation amplified by the second amplifying means tothe first applicator.

Also, in order to further increase flexibility of the apparatus theguiding means may comprise means for switching the electromagneticradiation amplified by the first amplifying means between the first andsecond applicator. Furthermore, the guiding means may comprise means forswitching the electromagnetic radiation amplified by the secondamplifying means between the first and second applicator.

The first and second applicators may be of various types. Preferable,the first and second applicators are selected from the group consistingof quasistatic, near field, surface field, single mode cavity and multimode cavity applicators.

The frequency of the electromagnetic radiation generated by the firstand second generating means may vary according to a first and secondcontrol signal, respectively. These first and second control signals maybe provided by the control means. Similarly, the amplification of thefirst and second amplifying means may vary in accordance with a firstand a second control signal, respectively. Also these signals may beprovided by the control means. The control means may comprise a generalpurpose computer. Such a general purpose computer may form part of aneural network.

The frequency of the electromagnetic radiation generated by the firstand second generating means is within the range 300 MHz-300 GHz, such aswithin the range 0.5-3 GHz or within the range 50-100 GHz.

In a second aspect, the present invention relates to a method forperforming a plurality of chemical reactions simultaneously orsequentially, said method comprising the steps of:

a) providing a first sample into a first applicator,

b) providing a second sample into a second applicator, and

c) applying electromagnetic radiation to the first and second samplessimultaneously or sequentially for a predetermined period of time, saidelectromagnetic radiation having a frequency in the range of 300 MHz-300GHz.

The electromagnetic radiation may be provided specifically andindependently to each of the samples. In addition, the appliedelectromagnetic radiation may comprise one or more pulses. The samplesmay be collected in sets comprising at least two holders. The sampleitself may be a PCR mixture. During exposure of a sample theelectromagnetic radiation may be applied in cycles of at least two stepswhere the sample is cooled at least during part of each cycle.

Preferably, the electromagnetic radiation is provided using an apparatusaccording to the first aspect of the present invention.

In a third aspect, the present invention relates to a method forperforming a chemical reaction, said method comprising the steps of:

a) providing a sample in an applicator,

b) applying electromagnetic radiation to the sample for a firstpredetermined period of time at a first level of power and varying thefrequency of the electromagnetic radiation between two predeterminedvalues and with a predetermined resolution, and determining a reflectionfactor of electromagnetic radiation from the sample at least some (two)of the frequencies covered by the range of the two predetermined valuesby determining the level of a feed-back signal, thereby obtaining afirst set of reflection factors,

c) changing the physical and/or chemical properties of the sample,

d) applying electromagnetic radiation to the applicator at a secondlevel of power and varying the frequency of the electromagneticradiation between two predetermined values and with a predeterminedresolution, the range defined by the predetermined values being includedin the range defined by the predetermined values in step b), anddetermining a reflection factor of electromagnetic radiation from thesample at least some (two) of the frequencies covered by the range ofthe two predetermined values by determining the level of the feed-backsignal, thereby obtaining a second set of reflection factors, and

e) repeating step c) and d) until the difference in reflection factorscalculated as the mathematical difference (subtraction) between thefrequencies associated with the first and second set of reflectionfactors is within a predetermined range.

Step c) may comprise applying electromagnetic radiation for heating thesample. Alternatively or in addition, the sample may also be cooledand/or a reagent may be added to the sample. Also, if the difference inreflection factors is within the predetermined range after the firstexecution of step c) and d), step e) will off course no longer apply.Furthermore, if the difference is close to being within thepredetermined range, it might not be economical to perform step e), andit may be omitted.

In a fourth aspect, the present invention relates to a method forperforming a chemical reaction, said method comprising the steps of:

a) providing a sample in an applicator,

b) applying electromagnetic radiation to the sample, the electromagneticradiation having a starting frequency,

c) varying the frequency of the applied electromagnetic radiationbetween two predetermined values and with a predetermined resolution,

d) determining a reflection factor of electromagnetic radiation from thesample by determining a level of a feed-back signal during at least partof the varying of the frequency of the electromagnetic radiation, and

e) determining, from the level of the feed-back signal, the frequency ofthe electromagnetic radiation where the reflection factor is within apredetermined range.

In a fifth aspect, the present invention relates to a method forperforming a chemical reaction, said method comprising the steps of:

a) providing a sample in an applicator,

b) applying electromagnetic radiation to the sample, the electromagneticradiation having a starting frequency,

c) varying the frequency of the electromagnetic radiation incrementallyaround the starting frequency,

d) determining a reflection factor of electromagnetic radiation from thesample by determining a level of a feed-back signal at the startingfrequency, at a frequency incrementally lower than the startingfrequency and at a frequency incrementally higher than the startingfrequency,

e) repeating step b) to d) until the reflection factor is minimum.

In a sixth aspect, the present invention relates to a method forperforming a chemical reaction, said method comprising the steps of:

a) providing a sample in an applicator,

b) applying electromagnetic radiation to the sample, the electromagneticradiation having a starting frequency,

c) varying the frequency of the electromagnetic radiation incrementallyaround the starting frequency,

d) determining a reflection factor of electromagnetic radiation from thesample by determining a level of a feed-back signal at the startingfrequency, at a frequency incrementally lower than the startingfrequency and a frequency incrementally higher than the startingfrequency,

e) comparing the determined reflection factor with a predeterminedreflection factor,

f) adjusting the starting frequency to a frequency so that thedetermined reflection factor is within a predetermined range around thepredetermined reflection factor, and

g) repeating step c) to f) as often as desirable.

The starting frequency may be in the range of 300 MHz-300 GHz. Thepredetermined values between which the frequency of the electromagneticradiation may be varied are in the range of 300 MHz-300 GHz, such aswithin the range 0.5-3 GHz or within the range 50-100 GHz. Preferably,the reactions according the third, fourth, fifth and sixth are performedusing an apparatus according to first aspect of the present invention.

In a seventh aspect, the present invention relates to a method forperforming a chemical reaction, said method comprising the steps of:

a) providing a sample in an applicator,

b) applying electromagnetic radiation to the sample in form of a firstpulse with a predetermined shape and characterising a reflected pulsefrom the applicator by performing a mathematical operation so as toobtain a first reflected spectrum,

c) changing the physical and/or chemical properties of the sample,

d) applying electromagnetic radiation to the sample in form of a secondpulse with a predetermined shape and characterising a reflected pulsefrom the applicator by performing a mathematical operation so as toobtain a second reflected spectrum,

e) repeating step c) and d) until the difference between the first andsecond reflected spectra calculated as the mathematical difference(subtraction) between the first and second spectra is within apredetermined range.

If the difference in reflection factors is within the predeterminedrange after the first execution of step c) and d), step e) will offcourse no longer apply. Furthermore, if the difference is close to beingwithin the predetermined range, it might not be economical to performstep e), and it may be omitted. Preferably, the mathematical operationfor obtaining the first and second reflection spectra comprises FourierTransformation but alternative operations may also be applicable. Themethod according to the seventh aspect of the present invention may beperformed using an apparatus according the first aspect of the presentinvention.

In an eighth aspect, the present invention relates to the use of anapparatus according to the first aspect of the present invention forheating at least one sample comprising at least one organic compound.Each of the samples may further comprise one or more reagents andoptionally a catalyst. Preferable, the apparatus according the firstaspect of the present invention is configured to heat two or morereaction mixtures, such as PCR mixtures, simultaneously or sequentiallyor intermittently.

The frequency of the electromagnetic radiation, the level of irradiatedpower and the period of applying the electromagnetic radiation isdetermined by pre-set values for the chemical reaction in question, suchpre-set values being stored in a storage means associated with thecontrol means. Corresponding data of frequency and reflection factor maybe stored in a memory for further processing. Further processing may beperformed in a neural network.

In a ninth aspect, the present invention relates to a kit for chemicallyreacting chemical species with a reagent optionally under the action ofa catalyst, wherein the chemical reaction is performed in an apparatusaccording to the first aspect of the present invention, said kitcomprising:

a) a sample holder comprising at least one of the reagent and theoptional catalyst,

b) an electronic storage means comprising data concerning the chemicalreaction between the chemical species and the reagent under the optionalaction of the catalyst, said electronic storage means and apparatusbeing adapted for retrieving the data from the storage means andprocessing said data so as to control the application of anelectromagnetic radiation to said sample holder.

This aspect may further comprise instructions regarding addition of thechemical species to the sample holder.

FIG. 1 illustrates possible combinations of the three main modules in anapparatus according to the invention.

FIG. 2 illustrates an embodiment comprising of the apparatus accordingto the present invention.

FIG. 3 illustrates an applicator mounted in a microtiter plate.

FIG. 4 illustrates a microtiter plate with a microwave conductor mountedsymmetrical in the centre of four wells.

FIG. 5 illustrates a microtiter plate with transmitting type applicatorwith input and output parts 12 and 13.

FIG. 6 illustrates a microtiter plate with an individual antenna foreach sample well.

As mentioned above, the present invention provides an apparatus andmethods for performing chemical reactions, preferably chemical reactionsperformed in parallel. A particular interesting feature of the apparatusaccording to the invention is the use of semiconductor components in thesignal generator and amplification means. As will be clear from thefollowing, the semiconductor signal generator offers hitherto unrealisedadvantages in chemical synthesis and thus also in the methods accordingto the invention.

The main purpose of utilising microwaves or other electromagneticradiation in an apparatus and methods for performing chemical reactionsis to heat and/or catalyse reactions taking place in a sample exposed tomicrowave radiation. Preferably the sample is placed in a sample holderin the applicator of the apparatus.

Also, according to the apparatus and the method according to the presentinvention, the signal generator can be controlled in response to theactual level of signal energy supplied to—and/or absorbed in—theapplicator. This feedback makes it feasible to control e.g. thetemperature of the samples to a very high degree.

The term microwave is intended to mean electromagnetic radiation in thefrequency range 300 MHz-300 GHz. Preferably, the apparatus and methodsaccording to the invention are performed within the frequency range of500 MHz-300 GHz, preferably within the frequency range 500 MHz-30 GHzsuch as 500 MHz-10 GHz such as 2-30 GHz such as 300 MHz-4 GHz such as2-20 GHz such as 0.5-3 GHz or within the range 50-100 GHz.

FIG. 1 illustrates a preferred embodiment of an apparatus according tothe present invention. The number n of signal generators 28 that areseparately amplified by signal amplifiers 29 are connected to the numberm of separate applicators 24 through the distributing network 23,represented by the box in the centre. It is seen that all components areconnected to the power supply 44 and the controller 45. FIG. 1illustrates parallel processing of samples, and that generators andapplicators are preferably controlled in response to the coupling ofmicrowave energy in the distributing network, the applicator or thesample. It should be mentioned that each applicator 24 can contain oneor more samples.

If the average power to be delivered to each applicator 24 is less thanthe maximum output power of an amplifier 29, the number of applicators24 can exceed the number of generators 28 and amplifiers 29, hence n<m.If the average power to be delivered to each applicator 24 is largerthan the maximum output power of an amplifier 29, the power for eachapplicator can originate from several amplifiers. Hence the power outputfrom some amplifiers can be distributed to several differentapplicators. In this case the number of applicators 24 can be less thanthe number of generators 28 and amplifiers 29, hence n>m. This guidingand coupling of radiation between amplifiers and applicators isperformed by the distributing network 23. Each amplifier and applicatorcan also be coupled in pairs, that is n=m.

In the following, the individual components comprised in the apparatuswill be described in more detail, including some preferred features.

The generating means 28 and the amplifying means 29 are essentiallyconstituted by semi-conductor components in order to be able to generatea signal between 300 MHz and 300 GHz, several individual semiconductorbased generators may be needed.

The power of the generated signal varies continuously between 0 and 1 W.The signal generator is capable of driving a signal amplifier and/or apower amplifier. Furthermore the signal generator iscontrollable/programmable from the controller 45. The control functionsis in the form of controlling the amplitude, frequency, frequencybandwidth, signal form, pulse form or duration of the signal/pulse andany combinations of two or more functions at the same time.

Semiconductor based microwave generators and amplifiers provide avariety of advantages over conventional TWT's, gyrotrons and magnetrons.Examples of these advantages are:

Easy control of frequency and output power

Small physical dimensions

No high voltage required, which improves safety and reliability

No warm-up time, therefore immediately availability

No wear-out parts which significantly reduce cost maintenance andimprove apparatus uptime

Far higher MTBF and lower MTTR compared with TWT

Better gain curve flatness compared with TWT

Lower noise compared with TWT

The amplifying means 29 can comprise a signal amplifier 29 and a poweramplifier 30, as shown in FIG. 2. The signal amplifier 29 is asemiconductor-based device being adapted to amplify the signal from thesignal generator. The gain of the amplifying means is adjustable byvarying the level of a control signal. Thus the amplitude of the outputcan be selected by the operator.

The power amplifier 30 is provided for further amplifying the signalfrom the signal amplifier. The power amplifier is also asemiconductor-based device with an adjustable gain. The gain is variedby varying the level of a control signal.

The heating power applied to the applicator is preferably in the rangeof 1-2000 W depending on the sample size and the chemical reaction inquestion. Typical ranges are 1-300 W such as 5-50 W, 10-1000 W such as30-100 W, and 50-2000 W such as 100-1000 W.

The necessary power of an electromagnetic radiation used for monitoringor “scanning” (see below) is typically only a fraction of the powerneeded for heating. Typical ranges are 0.05-100 W such as 0.1-10 W. Thetime of application also varies depending on the sample, process and thechemical reaction in question. Typical reaction times are 0.1 sec to 2hours such as 0.2-500 sec or 0.5-100 sec.

The amplified signal from the amplifying means is distributed to one ormore applicators using a distributing network.

The distributing network can comprise many features. FIG. 2 shows anembodiment of the apparatus comprising a selection of these features.FIG. 2 is only an example illustrating how the different features can beimplemented, and the order of the features in FIG. 2 is not restrictive.The following features can be comprised in the distributing network:

-   -   circulators 31    -   bi-directional couplers 32    -   power meters 34-38    -   artificial loads 33    -   dividers 51    -   combiners 50    -   spectrum analysers

Some of these features will be described in the following with referenceto FIG. 2.

The circulator 31 prevents the reflected power from the microwaveapplicator 24 and the distribution network 23 from entering the poweramplifier 30. Instead the reflected power is directed to a dummy load 33optionally connected to a first power meter 34. Some semi-conductorbaser generators and amplifiers, e.g. Silicon-carbidegenerators/amplifiers, are not affected by backscattered microwaves, andthe circulator 31 is not necessary when such generators/amplifiers areutilised.

The circulator 31 is adapted to be operationally connected between theamplifying means and the distributing network, and has at least oneinput terminal, an output terminal and at least one combinedinput/output terminal. The input terminal is operationally connected tothe output terminal of the amplifying means and the combinedinput/output terminal is operationally connected to the distributionnetwork. Furthermore, the load 33 and first power meter 34 can beincorporated in the apparatus in connection with the circulator.

The distributing network can comprise a coupler, such as abi-directional coupler 32, said coupler comprising an input terminal, atleast two output terminals and a combined input/output terminal. Theinput terminal can be operationally connected to the output terminal ofthe circulator or amplifier and the output terminal is operationallyconnected to other parts of the distributing network.

The bi-directional coupler directs a fraction of the input and/or thereflected power to two power meters 35 and 36. The third power meter 36measures a portion of the power transmitted in the direction towards theapplicator(s), whereas the second power meter 35 measures a portion ofthe power transmitted in the opposite direction, i.e. away from theapplicator(s). The power determining means can provide signals to thecontroller 45.

The distribution network can also comprise combiners 50 and dividers 51in order to facilitate parallel processing. These can include switchesso that the structure of the network can be varied.

In general, the distribution network is provided for distributing theelectromagnetic radiation generated and amplified using thesemiconductor signal generator and the semiconductor amplifiersrespectively. The generated and amplified signal can be distributed to asingle or to a plurality of applicators.

An example of such a network is coaxial cables with dividers in order tosplit up the power/signal line in as many power/signal lines as neededto feed all the separate applicators. Alternative ways of accomplishinga distributing network are to use wave-guides, strip-lines etc. Thedistributing network can be an integral part of the applicator design aswill be showed in FIGS. 3, 4, 5 and 6.

Applicators such as 24 can be of various types. According to the presentinvention some features are preferably comprised in the applicator. Someof these preferred features will be described in the following withreference to FIG. 2. A more detailed description of a number of embodiedapplicators will be given later.

The minimum requirements of an applicator are:

a) an input terminal 12,

b) a sample holder 1, and

c) means for confining the microwave energy from to the input terminal12.

In order to control the operation of the signal generator and amplifierin response to the power absorbed in the sample (or reflected by theapplicator), some measure of the total power absorbed in—and reflectedby—the applicator has to be obtained.

In order to determine the absorbed amount of power (or energy) in thesample, the applicator can comprise means for determining theelectromagnetic field strength. The applicator can comprise an outputterminal operationally connected to a load 33 that absorbs the reflectedpower from the applicator. Furthermore, fourth power measuring means 37are operationally connected to the load 33 and the control means 45.Also, a loop antenna 13 can act as microwave receiving means. The loopantenna is connected to a fifth power measuring means 38 and the controlmeans 45.

The above mentioned load 33 and loop antenna 13 are used for monitoringand receiving the microwaves transmitted through the sample 1 bytransferring the energy to power meters 37 or 38. The difference betweenthe power irradiated at the sample and the power transmitted/reflectedby the sample, measured with respective power meters depending on theexact setup, indicates the sum of the energy losses in the system andenergy absorbed in the sample. The applicator can be calibrated bymeasuring the system losses of the unloaded applicator before the sampleis introduced into the applicator. The energy absorbed in the samplewill characterise the sample in terms of dielectric properties at agiven temperature and frequency. By scanning the frequency within agiven range, e.g. 1-4 GHz, and monitoring the signals from the load 33or receiving antenna 13 together with the reflected signal from 35, itwill be possible to follow the progress of a chemical reaction.

The applicator can also include sensors operationally connected to thecontroller in order to monitor and control the application of microwaveenergy to the sample or samples. Sensors for measuring any parametercharacterising the extent of the process or reaction, such as pressure,temperature, pH, and conductivity, during the heating (and anyintermediate non-heating phases thereof) can be comprised. One possibletemperature sensor for microwave cavities is described in WO 94/24532.The output from such sensors can also provide a measure of the amount ofpower absorbed in the sample.

Spectrum analysers can be connected to the power measuring means, andthe power measuring means can be frequency selective. If theelectromagnetic signal directed to the applicator is time dependent,e.g. pulsed, analysis of the time and frequency spectra of a pulseirradiated at the sample, and the reflected/transmitted signal, canyield valuable information of the sample. This analysis can compriseFourier transformation of the measured signals. This feature is notspecifically connected to the applicator, rather it is a combination ofmeasurements from power meters at different locations in the system,together with analysing means which can be comprised in the controller.

The applicator is preferably adjustable so that it can be tuned tosupport modes depending on the used frequency. It should be noted thatthe applicator can have a quasistatic, near field, surface field, singlemode cavity or multi mode cavity, as well as an open ended cavity. Theapplicator can be tuned to make its resonance frequency correspond tothe frequency of the signal connected to the input terminal 12, e.g. byadjusting certain geometrical parameters, such as a resonator rod, ofit.

The sample 1 can be placed directly in the applicator, but the sample istypically placed in an open or closed sample holder 2. Such sampleholder could be an integral part of the applicator or a separatereaction vessel of any material suitable for use in microwave heatingapplications. As will be known to the person skilled in the art, thematerial constituting the sample holder should preferably not absorb themicrowave energy. Various types of polymers and glasses can be used.Specifically, various types of trays, microtiter plates, etc. canpreferably be used when a plurality of samples is heated simultaneously.A plurality of sample holders can be assembled in a sample holder set,such set-up can generate a very even heating of all samplessimultaneously.

The sample holder can furthermore be provided with sample inlet andoutlet ports for sample transfer in and out of the applicator and thesample holder during or between the process steps or whole processes.

The free space in the applicator can be filled with an inert gas inorder to avoid reaction between gasses and the sample. It is howeverpreferred that the sample holder include a lid. It is preferred that theapplicator includes at least one inlet/outlet for providing an inertatmosphere to the space above the sample. Alternatively, the space abovethe sample is filled with a reactive gas, e.g. H.sub.2, which is usefulin hydrogenation reactions.

The applicator should preferably be able to sustain high internalpressure either formed by the chemical reaction or formed intentionallyto create a high-pressure atmosphere as a reaction parameter. Highinternal pressure is normally used as a method to increase thetemperature of the sample over the boiling point for the liquid phase.The pressure can be kept at a predetermined level or pre-set as a levelnot to be exceeded or fall below. A pressure system incorporates asafety valve function for protection of the pressurised components andpersonal safety.

Rapid cooling of samples can be a very practical feature, which can becomprised in the applicator. Normally, when cooling samples without anyuse of means for cooling, the time for the sample to reach ambienttemperature is usually quite long, leading to undesired side reactionsand other unwanted phenomena. A forced cooling can therefore be used tominimise the time it takes for the sample to reach a predeterminedtemperature. The cooling device can be of any sort e.g. circulating air,circulating water or other liquid cooling media, peltier elements, etc.The cooling device can also be used to control the temperature duringthe process cycle. One important application of the cooling device iswhere temperature cycling of the sample is desirable. A pre-programmedtemperature cycle is used to control the heating of the sample withmicrowaves and cooling of the sample by using the cooling device. Anexample of such an application is temperature cycling to perform the PCRreaction (Polymerase Chain Reaction).

The controller 45 has a central function as shown in FIG. 2. Thecontrolling device is a computer based system for controlling (run-timecontrol) and programming of the apparatus and all itsmodules/components.

The controller 45 might be connected to one or several PCs in a networkas a user interface and/or computing device for one or several microwaveapparatuses. In this way storage means for storing data and/or processeddata and/or data concerning predetermined process parameters becomeavailable.

The control signal provided to the generating means 28 by the controller45 varies according to a first function of the back-reflected ortransmitted signal from the applicator 24, said back-reflected ortransmitted signal being detected by one of the power measuring means34-38.

The control signal provided to the amplifying means 29 and 30 by thecontroller varies according to a second function of the back-reflectedor transmitted signal from the applicator, said back-reflected ortransmitted signal being detected by one of the power measuring means34-38.

The control signal provided to the generating means 28 determines theoutput frequency, the control signal provided to the amplification means29 and 30 determines the amplitude of the amplified signal. Theamplitude of the amplified signal can be varied as a function of time.

The control system has three different modes of operation:

1) heating mode

2) monitoring mode

3) programming mode

Operating the controller 45 in heating mode puts specific requirementsto the configuration of the controller. The controller is capable ofsetting and controlling the output power from the signal amplifier 29and the power amplifier 30. Furthermore, the controller is capable ofmodulating the signal generated by the signal generator 28 so as togenerate an output signal, which is a function of time such as arectangular or triangular wave form. In the same context, the dutycircle of the signal must be adjustable so as to reduce the power of thedelivered signal.

The above-mentioned feature is provided by applying a first controlsignal to the signal amplifier 29 and a second control signal to thepower amplifier 30.

Another feature, which has to be incorporated in the controller, is theability to control the output frequency of the signal generator. Alsothe settings relating to frequency scans, i.e. start frequency, stopfrequency, frequency resolution and scan time must be controllable fromthe controller. The starting frequency is in the range of 0.5-300 GHz,preferably in the range of 1-30 GHz. Predetermined values between whichthe frequency of the electromagnetic radiation is varied are in therange of 0.5-300 GHz, preferably in the range of 1-30 GHz. Furthermore,the process time for a complete process or parts of a process if itinvolves more than one step should be controllable.

Measuring the input power to the applicator by means of a power meter 36is accomplished, however, the optimal position of power meter 36 dependson the exact configuration of the distributing network. Likewise, thereflected power from the applicator is measured with power meters 34 or35 whereas 37 or 38 measures the power coupled out from the applicator.The power absorbed in the applicator can be measured by calibrating theapparatus with an empty cavity to measure the losses in the applicator.This calibration can be done within the frequency range where the sampleis to be processed. By subtracting the reflected power and the losspower of an empty applicator the absorbed power can be calculated.

The power signal measured by the power meters 34 to 38 are transmittedto the controller so as to be used for controlling the frequency of thesignal generator 28 and/or the gain in the signal amplifier 29 and/orthe power amplifier 30.

The controller 45 can also provide control signals for systemcomponents—such as directional couplers 32, circulators 31, etc. Thecontroller can provide other types of signal processing. The controllercan control and monitor sample parameters such as temperature, pressure,pH, conductivity, etc, using the previously mentioned sensors. Bycurrent measuring of such parameters, the controller can respond if aparameter reach a predetermined values. It is possible to set a maximumvalue not to be exceeded during the process and a minimum value not tofall below during the process.

Determining the coupling between the electromagnetic radiation and thesample and varying the frequency and power of the radiation isessential. Furthermore, the frequency of the electromagnetic radiationcan be changed in response to a change of the level of the feedbacksignal by more than a predetermined threshold value. Data concerning thefrequency and the coupling efficiency—measured as a reflectionfactor—between the electromagnetic radiation and sample 1, can be storedin a memory for further processing.

In the monitoring mode, a scan function is available that normalises thesignal from a first scan (gives a strait baseline), and detects thedifference from the normalised baseline during a number of subsequentscanning cycles. Tracking and locking to the frequency that givesmaximum power absorbed in the sample 1, (moving maxima) is anotheravailable feature. The frequency of the microwave generator 28 isadjustable to an extent of at least .+−0.30% around a centre frequency

When the apparatus operates in programming mode the possibility ofcreating, storing, retrieving and editing using an in-built high levelmethod programming language must be available for the operator. A methodis a pre-programmed sequence of events where every event has at leastone process as input. A process parameter is e.g. power, time pressureetc.

The apparatus can also comprise means of collecting and processing allprocess data and store and/or retrieve said data from an internal and/orexternal database.

By using an apparatus with said monitoring and controlling meanscombined with at least one of the following parameters to be variable:frequency, waveform, power, time, temperature, pressure, artificialatmosphere, it is possible to optimise and maintain these optimalconditions for said chemical reaction.

Referring again to FIG. 2, an apparatus for microwave assisted chemicaland biological reactions is illustrated. One of the main features of theapparatus aims at optimising the reaction conditions for said chemicalreaction. Another set of features of the apparatus aims at monitoringand controlling the optimised conditions for said chemical reaction. Yetanother set of features aiming at process data collection, dataprocessing, storing and retrieving data from an internal and/or anexternal database.

When two or more starting materials react chemically they are subject tochanges in their physical and chemical properties. These changes inproperties are usually temperature dependent. Chemical reactions areoften performed at elevated temperature to enhance the speed of thereaction or supply enough energy to initiate and maintain a reaction.The form of the supplied energy could be thermal radiation, ultrasound,microwaves etc. In the case of microwaves as supplied form of energy thetransferred energy into the reacting materials is dependent on thedielectric properties of the starting and formed materials during thechemical reaction. The dielectric properties are temperature dependentand will therefore vary during the chemical process. Changes indielectric properties will also take place due to forming of newmaterials in the chemical reaction. The dielectric properties ofmaterials are also known to change with the frequency.

In an apparatus with frequency tuning, an optimum of coupled energy intothe reaction will occur at a specific frequency. This frequency willchange according to the temperature in the reaction in accordance withthe dependence of the samples' permittivity ∈′ upon temperature.

The term “chemical reaction” is intended to mean any inorganic andorganic reaction involving the formation or breaking of a (covalent)bond between two atoms, as well as conformer reactions of clusters andlarge molecules. It should be understood that the term also includesreactions where enzymes are involved as catalysts, e.g. the polymerasechain reaction (PCR) and similar types of reactions. The chemicalreaction is preferably a reaction involving organic compounds, i.e. lowmolecular organic compounds and biological organic compounds (e.g.enzymes). It is furthermore preferred that a conversion of the chemicalconstitution of one or more organic compound takes place.

The chemical reactions are typically organic chemical reactions of whichvirtually all known reactions are applicable. Typical reactions typesare polymerisation/oligomerisation, esterification, decarboxylatio,esterification, hydrogenation, dehydrogenation, addition such as1,3-dipolar addition, oxidation, isomerisation, acylation, alkylation,amidation, arylation, Diels-Alder reactions such as maleinisation andfumarisation, epoxidation, formylation, hydrocarboxylation,hydroboration, halogenation, hydroxylation, hydrometallation, reduction,sulphonation, aminomethylation, ozonolysis, etc. It is believed that theapparatus and methods according to the invention are especially suitedfor reactions involving one or more catalysts and for asymmetric organicreactions.

The chemical reaction can take place in a suitable solvent or in neatform. When a solvent is used, it is preferred that the dissipationfactor (or loss tangent) is greater than about 0.04 at room temperature.Examples of suitable solvents areacetonitrile, DMF, DMSO, NMP, water,tert-butanol, EtOH, benzonitrile, ethylene glycol, acetone, THF. Thefrequency of the generated electromagnetic signal can be tuned toabsorption bands/peaks for the used solvent.

The chemical reactions typically involve a starting material (substrateor “chemical species”), a reagent and optionally a catalyst (e.g. anenzyme such as a thermostable DNA polymerase). The starting material canbe any chemical substance in any phase, solid phase, liquid phase or gasphase. Included in starting materials are all materials used for e.g.solid support of reactants in chemical reactions. Starting materialsalso includes all materials (chemical substances) formed under thechemical reaction and can be considered as new starting material for asubsequent chemical reaction during the same process or in a new processperformed in the same apparatus. Staring material or reagents can alsobe included in the gas phase of an artificial atmosphere. The finishedchemical product from a previous chemical reaction, performed in theapparatus, shall also be considered as starting material for asubsequent chemical reaction performed in the apparatus.

The applicator 24 comprises a cavity or cavities for applying microwaveenergy to one or more samples 1. It should be understood that thevarious types of cavities and arrangements of cavities representdifferent embodiments of the applicator in the apparatus according tothe present invention. As the apparatus in principle may involve anapplicator of any known type (although with different degrees ofsuccess), the present invention is not limited to the specificallymentioned variants. In the following, different embodiments showingdifferent applicator designs and degree of parallel processing isdescribed. These embodiments may serve as applicator 24 in relation toFIGS. 1 and 2.

FIG. 3 illustrates a number of cavities mounted in an array. This arraycan be, but is not limited to, a microtiter plate 4. Each cavity isdefined by a lid 6, a bottom plate 8 and an outer metal tube 17. Eachcavity comprises a sample holder 2, a resonator rod 16 for adjusting theresonance frequency of the cavity, input and output signal loop antennas18, and optionally a gas inlet/outlet 15. The microwaves are introducedinductively through loop antennas 18 as showed in FIG. 3, alternativelythey can be introduced capacitative via a distributing network feedingthe whole array. The sample is placed on the resonator rod 16 in theouter tube 17 of the cavity. The length of the resonator rod can beadjusted for changing the resonance frequency of the cavity. Allcomponents are electrical connected to each other to form a closedelectrical circuit. The cavity could be pressurised and put under anartificial atmosphere.

Another application is illustrated in FIGS. 4A and B where four samplewells 9 are assembled symmetrical in a sample holder set. A shieldingmetal cage 3 serving as walls in a cavity surrounds the four sampleholders. The microwave transmitting device 5 is placed in the centre ofthe space defined between the four individual sample holders and therebyirradiates the four samples 1 simultaneously. Thus, in the embodimentillustrated in FIG. 4, a number (4 in the example) of samples areprocessed in parallel. As illustrated in FIG. 4B, a plurality ofcavities can be arranged in an array similar to the array described inrelation to FIG. 3.

FIG. 5 illustrates a configuration where the transmitting or receivingdevices, 12 or 13 respectively, are mounted on the bottom-plate 8, andwhere these devices form an array. The lid 6 is mounted on top of theplate, and the receiving or transmitting device, 13 or 12, can bemounted on the lid. The bottom-plate or the lid can be, but is notlimited to, a microtiter plate. The bottom-plate 8 and the lid 6 definea cavity with a metal tube 3. A vial made of a suitable material (glassor a polymer, e.g. polystyrene) is inserted into the metal tube to serveas a sample holder 2. A cooling device can be attached at thebottom-plate. In order to dissipate the microwave energy not absorbed,the lid can include a microwave absorbing material. The cooling devicecan also be attached to the lid in order to take care of the dissipatedenergy. An inlet/outlet port 15 for artificial atmosphere can beattached to the lid and/or the bottom-plate. The reaction vessel can bepressurised by using the artificial atmosphere or internally generatedpressure from the chemical reaction. Field confinement can be achievedby using a high permittivity body at 12 or 13. Thereby the lid can beremoved and the applicator becomes an open-end applicator.

FIG. 6 illustrates a microtiter plate with an individual antenna 5 foreach sample well, where the antenna is immersed in the sample well.Sample wells are arranged in an array and a metal tube 3 surrounds eachwell as a shield. A glass or plastic sample holder 2 is typicallyinserted into the metal tube 3 to serve as a sample holder. As in thecase of the embodiments of FIGS. 3 and 5, each sample is processedindividually.

General guidelines and instructions for the work with microwaves and theconstructions of microwave cavities are, e.g., given in Gabriel, et al.,Chem. Soc. Rev, 1998, Vol. 27, pp 213-223 and in Microwave Engineering,Harvey (ed.), Academic Press, London 1963 (in particular Chapters 4-6).

The apparatus according to the invention is suited for heating at leastone reaction mixture (sample) comprising at least one organic compound.The reaction mixture or each of the reaction mixtures (samples) canfurther comprise one or more reagents and optionally a catalyst (e.g. anenzyme).

In a particularly interesting embodiment, the apparatus is adapted forheating two or more reaction mixtures simultaneously or sequentially orintermittently.

In one important embodiment of the present invention, a plurality ofchemical reactions are performed in parallel. This is realistic due tothe cost efficient construction of the apparatus according to theinvention. FIG. 1 illustrates the principles behind the parallelprocessing of a plurality of samples.

The present invention also provides a method of performing a pluralityof chemical reactions simultaneously or sequentially, according to thethird aspect of the present invention described earlier.

This and the following methods are all suitable performed by using theapparatus defined herein.

The fact that the electromagnetic radiation can be adapted to eachsample (e.g. with respect to frequency, heating time, power, pulsing ofthe signal, signal cycles, etc.) is important, e.g. in optimisingprocesses and in the construction of libraries of chemical compounds. Inthe latter case, any differences in reactivity within the variousreagents and various substrates (and enzymes) can be compensated for bythe apparatus. Thus, in a further embodiment of the present invention,the apparatus is used for preparing a combinatorial library of compounds(at least 4 compounds). Also, the apparatus and the method according tothe invention can be used to prepare a large number of compounds in aparallel process, where the compounds are not part of combinatoriallibrary, i.e. where the compounds do not share common structuralfeatures. This is possible in a parallel process since the apparatus iscapable of coupling the application of the electromagnetic radiation toeach sample independently. A further interesting variant is thecontinuous preparation of compounds by using a sample holder having asample inlet and a sample outlet. In this latter situation, a sample canbe introduced in a sample holder formed as a loop or spiral of a tube, arinsing solution is subsequently introduced through the sample inletthereby forcing the sample out of the sample holder through the sampleoutlet, and a new sample is subsequently introduced. Due to therelatively short reaction time under microwave heating conditions, alarge number of samples can be processed in parallel (several sampleholders) or sequentially (one sample holder).

The process parameters, i.e. with respect to the frequency and the powerof the electromagnetic radiation, are controlled by the controller (45).As should be understood from the above, the electromagnetic radiation ispreferably provided by a semiconductor based signal generator, inparticular by an apparatus as defined in the first aspect of the presentinvention. In certain applications, e.g. where a heating/cooling cycleis required, the electromagnetic radiation is preferably appliedintermittently. Alternatively, any cooling means can be activatedintermittently.

As mentioned above, the electromagnetic radiation is adaptedspecifically to each of the samples, i.e. for each sample/sample holderthe process parameters are independently selected. This means that eachof the samples are processed under different conditions, or that sets ofsamples are treated under substantially identical conditions butconditions different from other sets of samples, or that all samples aretreated under substantially identical conditions. In the event that aset of samples is treated under substantially identical conditions, itcan be advantageous to use an applicator essentially as illustrated inFIG. 4, where the sample holders are collected in sets consisting of twoor more sample holders (a set of four sample holders is shown in FIG.4). Such sample holder sets typically consist of 2-1000 sample holders,typically from 3-96 sample holders.

The apparatus will be able to generate data as an expression of theprogress and completion of a chemical reaction. Such data can be storedin a database operationally associated with the controller. Furthermorethe database might be provided with information regarding the productarising from the chemical reaction, e.g. purity, enantiomeric purity,yield, etc. In the event that a plurality of reaction mixtures areheated simultaneously in separate cavities under different conditions(e.g. conditions with respect to frequency, heating time, heatingcycles, heating power, concentration of reagent, substrate and anycatalyst, signal shape, reflected power, transmitted power, temperature,pressure, artificial atmosphere, type of sample vial, etc) orsubsequently in the same or separate cavities under differentconditions, such data will after proper analysis (e.g. automatedstatistical analysis) provide a unique possibility of optimising thereaction condition for subsequent similar chemical reactions. Theprocessed data can be analysed with a suitable analysing method andevaluated to find optimal parameter settings and conditions. The resultfrom the process can be processed by multi-variant data analysis foroptimisation.

Furthermore it will be possible to provide a set of suitable reactionconditions for subsequent reactions of the same type, e.g. substitutionreactions using a specific class of catalysts, Diels-Alder reactionsusing specific substrates, etc.

In a further prospect of the present invention, it is envisaged thatsuch data for optimal (or suitable) process parameters for a number ofstandard type reactions can be identified by the supplier of theapparatus and be provided together with the apparatus according to theinvention. Thus, in a preferred embodiment, the storage means associatedwith the controller includes a section designated for predeterminedprocess parameters. Such a section could be formed as a replaceablememory card (or a “Smart Card”) which can be updated regularly by thesupplier of the apparatus and provided to the user of the apparatus.

Consequently, the present invention also relates to a method and the useas above wherein the frequency of the electromagnetic radiation suppliedto the sample in the applicator, the level of transmitted power and theperiod of application of the electromagnetic radiation is determined bypre-set values for the chemical reaction in question, such pre-setvalues being stored in a storage means associated with the controller ofthe apparatus.

Thus, a further interesting aspect previously described as the ninthaspect, the present invention is a kit for chemically reacting achemical species with a reagent optionally under the action of acatalyst, where the chemical reaction is performed in an apparatus asdefined in the first aspect of the present invention.

In the ninth aspect, it should be understood that the sample holderprovided with the kit can comprise one or more necessary reagents and/orany suitable catalyst so that the user only needs to provide thechemical species to the sample holder. The solvent (if a solvent isnecessary or desirable) is preferably also provided with the kit so asto ensure that the reagent and catalyst will become fullydissolved/dispersed. Alternatively, the sample holder can contain thereagent and/or the catalyst in immobilised form so as to facilitate theisolation of the product of the chemical reactions.

The apparatus makes it possible to perform a number of other valuablemethods for performing chemical reactions. In one embodiment, theprogress of the reaction is simultaneously monitored by scanning thesample before (reference set of reflection factors) and afterapplication of the electromagnetic radiation. By comparing a set ofreflection factors after and before (reference set) heating, theprogress can be determined. Comparison of microwave signals between areference situation (empty applicator) and a situation where a sample isintroduces in an applicator is described in U.S. Pat. No. 5,521,360. Inrespect of the present invention, it is possible vary the processparameters by means of the controller (45) in response to the measuredsets of reflection factors. The sets of coupling efficiencies canpreferably be normalised and/or transposed before comparison.

Thus, the present invention provides a method for performing a chemicalreaction according to the third aspect of the present inventiondescribed earlier.

In one intriguing variant (the “biosensor” variant) of the above method,the first (reference) varying of the frequency (step (b)) (a “scan”) isperformed prior to introduction of chemical substance to the sample. Thesample can comprise an enzyme or a biomolecule or a cell, for which thechemical substance is a substrate or a ligand. The subsequent “scan” isthen performed and the difference in reflection factor is expected toreflect the interaction between the chemical substance and thecomponents of the sample. This embodiment can be an especiallyinteresting variant for studying the interaction between aligand/substrate and an enzyme. The heating (step (c)) is often omittedin this variant. Furthermore, repeating the steps will only be necessaryin order to study the mentioned interaction over time, otherwise onlycomparison of two sets of reflection factors will be necessary.

Furthermore, the present invention also provides a method foridentifying minimum reflection (or two or more minima) for applicationof electromagnetic radiation (especially where the predetermined rangecomprises the frequency that provides optimal coupling between theelectromagnetic radiation and the sample). I.e. the present inventionprovides a method of performing a chemical reaction according to thefifth aspect of the present invention described earlier.

The invention also provides a method for seeking for a frequencyrepresenting a local (or global) reflection factor while performing achemical reaction, i.e. a method of performing a chemical reactionaccording to the sixth aspect of the present invention describedearlier.

The invention furthermore provides a method for seeking for a frequencywhere the reflection factor has a predetermined level while performing achemical reaction, i.e. a method of performing a chemical reactionaccording to the seventh aspect of the present invention describedearlier.

In an especially interesting variant of the methods described herein,each sample comprises at least one enzyme and, further, each sample is aPCR mixture.

The PCR reaction is a particularly interesting application for theapparatus according to the sixth aspect of the present invention as theapparatus provides means for varying and pulsing the energy applied (andthereby the temperature of a PCR vial) accurately. Furthermore, theapparatus comprises means for controlling and monitoring the progress ofthe PCR reaction.

The PCR technique is generally described in U.S. Pat. No. 4,683,202 andU.S. Pat. No. 4,683,196. The use of microwave radiation for heating PCRmixtures is known, i.e. from WO 91/12888, WO 95/15671 and WO 98/06876,however processing by using the apparatus according to the presentinvention provides unprecedented advantages over the known systems.General guidelines for handling and processing PCR mixtures (e.g.temperature ranges and cycle numbers and times) can be found in WO98/06876. A typical example of a temperature cycle for a PCR is adenaturation heating step up to around 80-100 degree. C. (e.g. 0.5-3minutes), a cooling step where the mixture is brought to around20-40.degree. C. (e.g. 0.1 to 1 minute) and a polymerisation step ataround 55-75.degree. C. (e.g. for 1-5 minutes). A complete amplificationreaction typically involves 15-100 cycles, e.g. around 25-35 cycles.

With the present invention it is possible to control the application ofenergy very accurately and to apply the energy in controllable doses andto cool the samples very rapidly so as to reduce the cooling steps.Furthermore, it is also possible to monitor the progress of thereactions by applying a low intensity microwave signal to the reactionmixture, e.g., in each cooling step so as to determine the completion(relative to certain criteria) of the reactions. Thus, theelectromagnetic radiation is preferably applied in cycles of at leasttwo levels where the samples are cooled at least during a part of eachcycle. The at least two levels can represent the temperature levels of80-100.degree. C. and 55-75.degree. C. Typically, the cooling isinitiated in order to reach a temperature level of 20-40.degree. C. Thecooling can also be applied constantly (e.g. in the form of a cold block(bottom plate) in order to obtain a steeper cooling gradient.

1-15. (canceled)
 16. A method for performing a chemical reaction, saidmethod comprising: a) providing a sample in an applicator, b) applyingelectromagnetic radiation to the sample for a first period of time at afirst level of power and varying the frequency of the electromagneticradiation between two values and with a given resolution, anddetermining a reflection factor of electromagnetic radiation from thesample at least two of the frequencies covered by the range of the twovalues by determining the level of a feedback signal, thereby obtaininga first set of reflection factors, c) changing at least one of thephysical and chemical properties of the sample, d) applyingelectromagnetic radiation to the applicator at a second level of powerand varying the frequency of the electromagnetic radiation between twovalues and with a given resolution, the range defined by the valuesbeing included in the range defined by the values in step b), anddetermining a reflection factor of electromagnetic radiation from thesample at least two of the frequencies covered by the range of the twovalues by determining the level of the feed-back signal, therebyobtaining a second set of reflection factors, and e) repeating step c)and d) until the difference in reflection factors calculated as themathematical difference between the frequencies associated with thefirst and second set of reflection factors is within a given range. 17.A method for performing a chemical reaction, said method comprising: a)providing a sample in an applicator, b) applying electromagneticradiation to the sample, the electromagnetic radiation having a startingfrequency, c) varying the frequency of the applied electromagneticradiation between two values and with a given resolution, d) determininga reflection factor of electromagnetic radiation from the sample bydetermining a level of a feed-back signal during at least part of thevarying of the frequency of the electromagnetic radiation, and e)determining, from the level of the feed-back signal, the frequency ofthe electromagnetic radiation wherein the reflection factor is within agiven range.
 18. A method for performing a chemical reaction, saidmethod comprising: a) providing a sample in an applicator, b) applyingelectromagnetic radiation to the sample, the electromagnetic radiationhaving a starting frequency, c) varying the frequency of theelectromagnetic radiation incrementally around the starting frequency,d) determining a reflection factor of electromagnetic radiation from thesample by determining a level of a feed-back signal at the startingfrequency, at frequency incrementally lower than the starting frequencyand at a frequency incrementally higher than the starting frequency, e)repeating step b) and d) until the reflection factor is at a minimum.19. A method for performing a chemical reaction, said method comprising:a) providing a sample in an applicator, b) applying electromagneticradiation to the sample, the electromagnetic radiation having a startingfrequency, c) varying the frequency of the electromagnetic radiationincrementally around the starting frequency, d) determining a reflectionfactor of electromagnetic radiation from the sample by determining alevel of a feed-back signal at the starting frequency, at a frequencyincrementally lower than the starting frequency and a frequencyincremental higher than the starting frequency, e) comparing thedetermined reflection factor with a reflection factor, f) adjusting thestarting frequency to a frequency so that the determined reflectionfactor is within a range around the reflection factor, and g) repeatingstep c) to f) as often as desirable.
 20. A method according to claim 16,wherein the starting frequency is in the range of 300 MHz-300 GHz.
 21. Amethod according to claim 17, wherein the starting frequency is in therange of 300 MHz-300 GHz.
 22. A method according to claim 18, whereinthe starting frequency is in the range of 300 MHz-300 GHz.
 23. A methodaccording to claim 19, wherein the starting frequency is in the range of300 MHz-300 GHz.
 24. A method according to claim 16, wherein the valuesbetween which the frequency of the electromagnetic field is varied arein the range of 300 MHz-300 GHz, such as within the range 0.5-3 GHz orwithin the range 50-100 GHz.
 25. A method according to claim 17, whereinthe values between which the frequency of the electromagnetic field isvaried are in the range of 300 MHz-300 GHz, such as within the range0.5-3 GHz or within the range 50-100 GHz.
 26. A method according toclaim 18, wherein the values between which the frequency of theelectromagnetic field is varied are in the range of 300 MHz-300 GHz,such as within the range 0.5-3 GHz or within the range 50-100 GHz.
 27. Amethod according to claim 19, wherein the values between which thefrequency of the electromagnetic field is varied are in the range of 300MHz-300 GHz, such as within the range 0.5-3 GHz or within the range50-100 GHz.
 28. A method according to claim 20, wherein the valuesbetween which the frequency of the electromagnetic field is varied arein the range of 300 MHz-300 GHz, such as within the range 0.5-3 GHz orwithin the range 50-100 GHz.
 29. A method according to claim 16, whereinthe reaction is conducted in an apparatus according to an apparatus forproviding electromagnetic radiation comprising: a) at least onegenerator for generating waves of electromagnetic radiation, each ofsaid at lest one generators being capable of generating electromagneticradiation at least one frequency, b) at least one guide for guiding atleast part of a generated wave of electromagnetic radiation to at leastone applicator, and c) a controller for individually controlling each ofsaid at least one generators in response to a control signal, thecontrol signal reflecting the status of a sample in on at least oneapplicators.
 30. A method according to claim 17, wherein the reaction isconducted in an apparatus according to an apparatus for providingelectromagnetic radiation comprising: a) at least one generator forgenerating waves of electromagnetic radiation, each of said at lest onegenerators being capable of generating electromagnetic radiation atleast one frequency, b) at least one guide for guiding at least part ofa generated wave of electromagnetic radiation to at least oneapplicator, and c) a controller for individually controlling each ofsaid at least one generators in response to a control signal, thecontrol signal reflecting the status of a sample in on at least oneapplicators.
 31. A method according to claim 18, wherein the reaction isconducted in an apparatus for providing electromagnetic radiationcomprising: a) at least one generator for generating waves ofelectromagnetic radiation, each of said at lest one generators beingcapable of generating electromagnetic radiation at least one frequency,b) at least one guide for guiding at least part of a generated wave ofelectromagnetic radiation to at least one applicator, and c) acontroller for individually controlling each of said at least onegenerators in response to a control signal, the control signalreflecting the status of a sample in on at least one applicators.
 32. Amethod according to claim 19, wherein the reaction is conducted in anapparatus for providing electromagnetic radiation comprising: a) atleast one generator for generating waves of electromagnetic radiation,each of said at lest one generators being capable of generatingelectromagnetic radiation at least one frequency, b) at least one guidefor guiding at least part of a generated wave of electromagneticradiation to at least one applicator, and c) a controller forindividually controlling each of said at least one generators inresponse to a control signal, the control signal reflecting the statusof a sample in on at least one applicators.
 33. A method according toclaim 20, wherein the reaction is conducted in an apparatus forproviding electromagnetic radiation comprising: a) at least onegenerator for generating waves of electromagnetic radiation, each ofsaid at lest one generators being capable of generating electromagneticradiation at least one frequency, b) at least one guide for guiding atleast part of a generated wave of electromagnetic radiation to at leastone applicator, and c) a controller for individually controlling each ofsaid at least one generators in response to a control signal, thecontrol signal reflecting the status of a sample in on at least oneapplicators.
 34. A method according to claim 24, wherein the reaction isconducted in an apparatus for providing electromagnetic radiationcomprising: a) at least one generator for generating waves ofelectromagnetic radiation, each of said at lest one generators beingcapable of generating electromagnetic radiation at least one frequency,b) at least one guide for guiding at least part of a generated wave ofelectromagnetic radiation to at least one applicator, and c) acontroller for individually controlling each of said at least onegenerators in response to a control signal, the control signalreflecting the status of a sample in on at least one applicators. 35-43.(canceled)